Bone Abstracts

Background: We have previously shown that MLO-Y4 osteocytes express a number of P2 receptors, respond to a broad range of nucleotides (e.g. UTP) by increasing intracellular calcium concentration and release ATP upon both mechanical and UTP stimulation. The aim of this study therefore is to investigate how the osteocytes release ATP and whether there is a difference in release pathway depending on the type of stimulus.

Methods: ATP release was investigated in vitro in MLO-Y4 osteocytes by measuring real-time luciferase-generated luminescence using the ATP Bioluminescence Kit HSII (Roche) and a NOVOstar luminometer (BMG Labtech). Mechanical stimulation was applied on the cells by injecting liquid into the wells at 310 μl/s. UTP stimulation was applied by injecting 10 μM UTP into the wells using the lowest speed level, 100 μl/s. The involvement of hemi-channels and vesicles in ATP release was studied by adding pharmacological inhibitors.

Results: It was found that mechanically-induced ATP release was significantly reduced by up to 50% when hemi-channels were blocked by 35–75 μM carbenoxolone (P≤0.05). In contrast, UTP-stimulated ATP release was significantly increased more than fivefold by 75 μM carbenoxolone (P≤0.05) and this effect was confirmed when using 10–200 μM pannexin-1 blocking peptide (P≤0.05). In addition to this, both mechanically and UTP-induced ATP release could be reduced by up to 50% when blocking the vacuolar H⁺-pump using 0.5–1 μM bafilomycin A1 (P≤0.05).

Conclusion: Results indicate that ATP signalling in the osteocyte network can be induced by both mechanical stimulation and P2 receptor activation. Mechanically-induced ATP release is indicated to occur via both hemi-channel and vesicular pathways, while UTP-induced ATP release at least in part occurs via a vesicular release pathway. The elevating effect of carbenoxolone and pannexin-1 blocking peptide on UTP evoked ATP release should be studied further.